Hepatitis C virus(HCV) is a primary cause of viral hepatitis which progresses into cirrhosis or hepatocellular carcinoma, and it has been reported that about 70 to 80% of hepatitis caused by blood transfusion is due to said virus(Alter, H. J., et al., Lancet, 2, 838-841(1975); and Dienstag, J. L., et al., Seminar Liver Dis., 6, 67-81(1986)). Said virus is one of RNA viruses consisting of one positive RNA strand and produces a polyprotein precursor from an open reading frame(ORF) of the strand(Choo, Q. L., et al., Science, 244, 359-362(1989); and, Choo, Q. L., et al., Proc. Natl. Acad. Sci. USA, 88, 2451-2455(1991)).
The gene structure of hepatitis C virus is similar to that of flavivirus or pestivirus(Miller, R. H., et al., Proc. Natl. Acad. Sci. USA, 87, 2057-2061(1990); and, Muraiso, K., et al., Biochem. Biophys. Res. Commun., 172, 511-516(1991)), and on the basis of said relationship it is presumed that the polyprotein of hepatitis C virus consists of, from N-terminal to C-terminal, core-envelope 1(E1)-envelope 2/non-structural 1 protein(E2/NS1)-non-structural 2 protein(NS2)-non-structural 3 protein(NS3)-non-structural 4 protein(NS4)-non-structural 5 protein(NS5)(Choo, Q. L., et al., Proc. Natl. Acad. Sci. USA, 88, 2451-2455(1991); Takamizawa, A., et al., J. Virol., 65, 1105-1113(1991); and Kato, N., et al., Proc. Natl. Acad. Sci. USA, 87, 6524-6528(1990)).
The infection of hepatitis C virus can be diagnosed by detecting hepatitis C viral RNA directly from a blood sample by using polymerase chain reaction(PCR)(Hosoda, K., et al., Hepatology, 15, 777-781(1992); Abe, K., et al., Hepatology, 15, 690-695(1992); and, Alter, H. J., Annals of Internal Medicine, 115, 644-649(1991), whereby the viral RNA can be detected rather early, i.e., within 1 to 2 weeks from the infection; however, such method entails high cost and long time due to the need to analyze numerous samples. Another diagnostic method is to detect antibodies against hepatitis C virus present in the serum sample, e.g., by an enzyme-linked immunoassay using C100-3 protein(see Houghton et al., PCT WO 89/04669; WO 90/11089). Kuo et al. disclosed in Science 244, 362-384(1989) that more than 70% of patients with post-transfusion hepatitis have antibodies against the C100-3 protein.
However, said C100-3 antigen used as an active ingredient for the diagnostic agents reacts only to the antibodies of patients with chronic hepatitis C, not with those of patients with acute hepatitis C at its early stage since the antibodies are not generally produced until 4 to 6 months after the HCV infection. As a result, it often exhibits a false negative during the early stage of the disease(Alber, H. J., et al., N. Engl. J. Med., 321, 1494-1500(1989); Myamura, T., et al., Proc. Natl. Acad. Sci. USA, 87, 983-987(1990)); and, further, it often exhibits false positive results in a considerable proportion in the case of hepatitis caused by the autoimmune disease of the patients and not by HCV(McFarlane, I. G., et al., Lancet, 335, 754-757(1990)).
Okamoto et al. disclosed the nucleotide sequences of the cDNA clones including the 5'-terminal region and structural genes encoding the core protein and envelope protein by using the HCV taken from the serum collected from Japanese hepatitis C patients, and compared said sequences with those of HCV extracted from the serum of chimpanzee which was prepared by Chiron Co. in the U.S. From that result, Okamoto et al. discovered the existence of a subspecific hepatitis C virus and the specificity of the antigens derived from Japanese type HCV for preparing vaccines and diagnostic agents against Japanese type HCV(Jpn. J. Exp. Med., 60, 167-177(1990)). Harada et al. further reported in J. Virol., 65, 3015(1991) that, when the core protein encoded in 5'-terminal portion of the structural gene was used as an antigen for diagnosing anti-HCV antibodies which may be present in the samples taken from putative patients, the antibodies could be detected 6 to 8 weeks earlier than the case of using C100-3 protein.
Further, Choo et al. disclosed an improved diagnostic method using a core protein expressed from a core structural gene and C33C protein expressed from NS3 gene(Br. Med. Bull., 46, 423-441(1990)); and, Okamoto et al. employed synthetic polypeptides synthesized by using the nucleotide sequences of a part of core structural gene to diagnose hepatitis C. UBI Co. of the U.S. developed another diagnostic method wherein synthetic polypeptides consisting of 15 to 65 amino acids encoded in core structural gene were employed as antigens for detecting anti-HCV antibodies(wang, C. Y., EP 442394(1991); Hosein, B., et al., Proc. Natl. Acad. Sci., 88, 3647-3651(1991). In addition, Ortho Diagnostic Systems Inc. of the U.S. reported a second generation diagnostic agent having improved sensitivity for anti-HCV antibodies which was prepared by adding core antigen C22-3, NS3 partial protein C33C and NS4 partial protein C200 to the pre-existing first generation diagnostic agent(McHutchison, J. G., et al., Hepatology, 15, 19-25(1992)); and Alter describes that it it is possible to detect anti-HCV antibodies from a serum taken from an HCV patient 15 to 20 weeks after the infection by HCV (Annals of Internal Medicine, 115, 644-649(1991)).
The present inventors also disclosed an intrinsic gene structure of Korean type hepatitis C virus(KHCV) different from the American type or Japanese type HCVs; expressed KHCV UBCORE14 protein from KHCV CORE gene, KHCV UB897 protein from KHCV NS3 gene, KHCV 403 protein from KHCV NS5 gene and envelope proteins from KHCV envelope gene in recombinant yeast or E. coli cells; confirmed the immunospecificity of the above expressed proteins; reported the process for purifying said proteins; and developed an improved diagnostic method to detect anti-HCV antibodies from a serum taken from a hepatitis C patient with the KHCV antigenic proteins by employing enzyme-linked immunosorbent assay(ELISA) (see Korean Patent Publication No. 93-683)
The present inventors have endeavored to develop HCV diagnostic agents with an improved accuracy and speed over the above agents As a result, there have been unexpectedly discovered several HCV epitopes which react with the antibodies against HCV with a greater sensitivity and accuracy.